Project Summary A differentiated, mature oocyte is ?activated? to become totipotent and to begin embryo development through an increase in its cytoplasmic free Ca2+ levels. In mammals, this increase is initially induced by the fertilizing sperm, which stimulates release of Ca2+ from internal stores. However, recent studies have shown that subsequent Ca2+ oscillations that are essential for mammalian egg activation require influx of external Ca2+ from the environment into the oocyte, through as yet unknown channels and mechanisms. Although Drosophila had not been considered a good model for these processes because its eggs activate prior to fertilization, our recent work has shown that Drosophila egg activation also involves a local Ca2+ influx. This influx is followed by a wave of increased Ca2+ that sweeps through the egg cytoplasm, using mechanisms like those used by mammalian oocytes. We showed that the initial Ca2+ influx in Drosophila occurs at the oocyte poles, through mechanosensitive channels ? most likely conserved channels in the TRP family. We propose to establish Drosophila as a new model for the Ca2+-based phase of egg activation. We will use this model to identify the channels that mediate sperm-independent Ca2+ influx and the conditions that cause Ca2+ channels to be open only at the egg poles. We will also determine whether the increased Ca2+ mediates the changes to the oocyte phosphoproteome that normally accompany egg activation, and whether the Ca2+ signal transducer calcineurin is required for these phosphoproteome changes. We expect the results of our studies to be generalizable to mechanisms in mammalian egg activation. Moreover, conserved proteins with Ca2+- dependent phosphomodifications will be candidates for future testing as markers for assessing human ART conditions, oocyte quality and aiding the diagnosis of infertilities that cannot respond to current ART methods.